Bacterial pathogens seek, tolerate and escape different environments by changing swimming direction (chemotaxis), surface motility, gene expression, biofilm formation, and the course of infection. Responses are species-specific but all are modulated by chemosensory systems composed of sensory receptors (chemoreceptors) and effector proteins. One such chemosensory system is regulated by a chemoreceptor called Aer2, which senses O2 via a PAS sensing domain that uses novel methods to coordinate heme and stabilize O2-binding. PAS signals are transduced directly through contiguous poly-HAMP domains. Aer2- regulated systems occur in a number of pathogens, including Pseudomonas aeruginosa, where the system is called Che2. P. aeruginosa is an important opportunistic pathogen, a significant cause of hospital-acquired infection, and a major cause of both morbidity and mortality in cystic fibrosis patients. The long-term goals of this project are to characterize the Aer2-regulated Che2 pathway in P. aeruginosa, from gas sensing to cellular response, and to determine the exact role of that response during infection. These goals align with the NIGMS mission to support basic research that increases our understanding of biological processes and lays a foundation for medical advances. In this initial project period, Aer2 sensing, signaling, an output responses will be characterized. Experiments in Specific Aim 1 will define unique Aer2 sensing and signaling mechanisms. A combination of mutagenesis, behavioral assays and ligand affinity experiments will be used to characterize Aer2 PAS sensing and signal initiation. To determine whether current, non-physiologic structures represent conformational changes that occur in vivo, PAS structures will be solved in both signal-on and signal-off states. Postulated PAS dimer-to-monomer transitions during Aer2 signaling will be assessed by disulfide crosslinking, surface accessibility, bacterial two-hybrid and size exclusion chromatography. To assess HAMP conformational inversions along the Aer2 poly-HAMP chain, signaling defects will be transposed among HAMP domains, and evaluated both behaviorally and for their influence on PAS quaternary structure. Experiments in Specific Aim 2 will assess the virulence of Aer2 and Che2 mutants in an in vivo Caenorhabditis elegans slow-kill assay. Effects on P. aeruginosa virulence will be compared with in vitro phosphorylation assays using Che2 protein components. The role of the Che2 chemosensory system in P. aeruginosa will be uncovered by identifying downstream interacting partners of the Che2 effector protein, CheY2; candidates will be isolated by in vitro pull-down assays, and in vivo bacterial two-hybrid assays with a cloned P. aeruginosa genomic library. Overall, these studies will use an experimentally accessible system to impact our understanding of how Aer2 receptors sense and signal; it will also provide insights into PAS/poly-HAMP signaling mechanisms that will be applicable to many other proteins. Lastly, this study will help define the role of Aer2-regulated chemosensory systems and clarify how that role impacts bacterial pathogenesis.